Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

Abstract

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases -the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time.

Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library

“…Our library is a multipurpose tool that can interact with enzymes according to the considerable differences in their structures. The inclusion of both natural and unnatural amino acids was previously reported to be useful for distinguishing between even very closely related enzymes [5,23,32,33]. Comparing the substrate specificity of individual enzymes shows their distinct activity against both natural and unnatural amino acids.…”

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease

“…Screening of Combinatorial Peptide Libraries-Enzyme activity toward libraries was measured as a relative fluorescence units per second using a plate reader (Molecular Devices) according to the previously described method (28,29). Kinetic analysis was performed at 30°C in 20 mM MES, 2 mM DTT, 5% glycerol, 500 mM NaCl buffer.…”

“…Synthesis of Optimized Peptide Substrates-Substrates were synthesized according to the previously described method (28). 1 eq of Fmoc-Rink Amide resin (0.7 mmol/g, 200 -300 mesh) was swollen for 30 min in DCM in a glass peptide synthesis vessel.…”

“…In the first step Fmoc-ACC-OH fluorophore was attached to 6 g of Rink Amide resin according to the procedure described elsewhere (28). After the Fmoc group deprotection (20% piperidine in N,N-dimethylformamide (DMF)), Fmoc-Asp(tBu)-OH (2.5 eq) was attached to the NH 2 -ACC-resin using 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo [4,5]pyridinium 3-oxide hexafluorophosphate (HATU, 2.5 eq) as a coupling agent and 2,4,6-trimethylpyridine (collidine, 2.5 eq) as a base.…”

Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi

“…The expanded P2 sublibrary, which additionally contained several unnatural amino acids, enabled generation of selective substrates, with which we were able to distinguish between the activities of two closely related homologues human Atg4B and T. cruzi Atg4.2. The use of unnatural amino acids has been successfully applied to identify substantially improved substrates of several other proteases including human cathepsin C (65), neutrophil elastase (41), and caspases (66). Using this approach, we were able to identify Ac-KKChaG-AFC as an optimal substrate distinguishing TcAtg4.2 from HsAtg4B.…”